Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Abstract

Tungsten microprojectiles coated with nucleic acid and accelerated to velocities of approximately 500 m/s, can penetrate living cells and tissues with consequent expression of the introduced genes (Klein et al. 1987). Saccharomyces cerevisiae is used here as a model system to define the basic parameters governing the biolistic (biological-ballistic) delivery of DNA into cells. Among the physical factors affecting the efficiency of the process in yeast are the microprojectile's constitution, size, concentration and amount, and the procedure used for binding DNA to it. The biological parameters that affect the process include the cell's genotype, growth phase, plating density, and the osmotic composition of the medium during bombardment. By optimizing these physical and biological parameters, rates of transformation between 10^-5 and 10^-4 were achieved. Stable nuclear transformants result primarily from penetration of single particles of 0.5–0.65 μm in diameter, delivering on average 10–30 biologically active plasmids into the cell. The tungsten particles detectably increase the buoyant density of the transformants' progenitors.